A Basic Sequence in STIM1 Promotes Ca2+ Influx by Interacting with the C-Terminal Acidic Coiled Coil of Orai1

Store-operated Ca2+ entry (SOCE) is a ubiquitous signaling process in eukaryotic cells in which the endoplasmic reticulum (ER)-localized Ca2+ sensor, STIM1, activates the plasma membrane-localized Ca2+ release-activated Ca2+ (CRAC) channel, Orai1, in response to emptying of ER Ca2+ stores, In efforts to understand this activation mechanism, we recently idetiltified an acidic coiled-coil region in the C-terminus of Orai1 that contributes to physical association between these two proteins, its measured by fluorescence resonance energy transfer, and is necessary for Ca2+ influx, as measured by an intracellular Ca2+ indicator. Here, we present evidence that a positively charged sequence of STIM1 in its CRAC channel activating domain, human residues 384-386, is necessary for activation of SOCE, most likely because this sequence interacts directly with the acidic coiled coil of Orai1 to gate Ca2+ influx. We find that mutation to remove positive charges in these residues in STIM1 prevents its stimulated association with wild-type Orai1. However, association does occur between this mutant STIM1 and Orai1 that is mutated to remove negative charges in its C-terminal coiled coil, indicating that other structural features are sufficient for this interaction. Despite this physical association, we find that thapsigargin fails to activate SOCE following coexpression of mutant STIM1 with either wild type or mutant Orai1, implicating STIM1 residues 384-386 in transmission of the Ca2+ gating signal to Orai1 following store depletion.